Overview of Program Project: This is a resubmission of a renewal of a Program Project Grant (PPG) application to study the mucosal barrier in infection and inflammation. Ninety-five percent of infectious agents enter through exposed mucosal surfaces, such as the respiratory, gastrointestinal, mammary and urogenital tracts. Mucosal infections include bacterial and viral pneumonia, SARS, TB, AIDS and other sexually transmitted diseases, numerous opportunistic, emerging and re-emerging infections, and biological warfare/terrorist agents. Mucosal surfaces are lined by epithelial cells, usually in a monolayer. The epithelial cell layer is the principal barrier to entry of infectious agents, allergens and other noxious antigens. Fundamentally, the epithelial layer is the most basic component of the innate mucosal immune system. In this Program Project we focus on two broad and inter-related areas of how the epithelial layer performs its functions in mucosal immune protection, with a particular emphasis on pulmonary epithelium. Projects 1 (Mostov), 2 (Engel) and 3 (Rosen) study mechanisms of epithelial wound healing, the roles of sulfs (sulfatases which modify heparan sulfate proteoglycans) in epithelial response to injury and the interaction of polarized cells with a bacterial pathogen. Project 4 (Werb) is focused on how inflammatory cells cross the epithelial barrier to enter the lumen of the organ. Project 1 uses two novel three dimensional cell culture models of epithelial wound healing. Project 2 focuses on the opportunistic pathogen, Pseudomonas aeruginosa, as a model for epithelial pathogen interaction. Project 3 examines how sulfs participate in epithelial wound healing. Project 4 focuses on the role of proteases and sulfs in regulating inflammatory cell recruitment and migration in mucosal tissues. Core A supports the Program Project administratively. Core B (Matthay) provides primary human and rodent lung epithelial cells in monolayer cultures, three-dimensional models and lung slices, as well as functional studies in mice. Core C has an array of four microscopes suitable for three- and four-dimensional analysis of cell dynamics for in vitro studies as well as experiments in living, anesthetized mice. Most infectious agents enter the body through exposed mucosal surfaces. These surfaces are lined by epithelial cells, most commonly in a single layer. We are studying how this epithelial barrier protects us against infection. PROJECT 1: Epithelial wound healing in 3 dimensions (Mostov, K) Project 1 Description (provided by applicant): Greater than 95% of infectious agents enter through exposed mucosal surfaces, such as the respiratory, gastrointestinal and genitourinary tracts. These include HIV, sexually transmitted diseases, numerous opportunistic infections, TB, many emerging and re-emerging infections, and biological warfare/terrorist agents, such as anthrax, Yersinia pestis and smallpox. Most mucosal surfaces are lined by a monolayer of polarized epithelial cells, which forms the principal barrier to entry by infectious agents. In essence, the epithelial layer can be considered the most basic component of the innate mucosal immune system. Some pathogens cross the epithelial layer by disrupting it. Other pathogens exploit disruptions in the monolayer, which can be caused by tissue injury secondary to inflammation, trauma, or may result from cell death or division within the monolayer. To maintain their function as a barrier to infection, epithelial tissues have developed efficient wound healing mechanisms. Wound healing is central to mucosal defense against infection. The epithelial barrier must be restored as quickly as possible, to minimize the opportunity for entry of infectious agents. Some infectious agents, such as Pseudomonas aeruginosa, not only exploit pre-existing wounds, but also impede the wound healing process. We are studying epithelial wound healing by growing epithelial cells in three-dimensional cultures of extracellular matrix, which causes the cells to more closely resemble in vivo conditions. In Aim 1 we are using a system of human primary lung alveolar type II cells, which form alveolar-like cysts, as a model to study response to acute lung injury/acute respiratory distress syndrome. In Aim 2, we are using a three-dimensional system of a well-differentiated human airway cell line, which forms cysts and tubules lined by pseudostratified epithelium, as a model to study airway response to injury. In Aim 3 we are studying the roles of matrix metalloproteinases and sulfatases (enzymes that remove 6-O-sulfate groups from heparan sulfate proteoglycans) in our three dimensional culture systems. This work will be in collaboration with Projects 2, 3 and 4, and supported by Cores B and C. Most infectious agents enter through the layer of cells that lines internal organs, such as the lung. Injuries to this cell layer make it much easier for pathogens to enter and we are studying how the cell layer heals itself.